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Theory predicts that threatened species living in small populations will experience high levels of inbreeding that will increase their genetic load, but recent work suggests that the impact of load may be minimized by purging resulting from long‐term population bottlenecks. Empirical studies that examine this idea using genome‐wide estimates of inbreeding and genetic load in threatened species are limited. Here we use individual genome resequencing data to compare levels of inbreeding, levels of genetic load (estimated as mutation load) and population history in threatened Eastern massasauga rattlesnakes (Sistrurus catenatus), which exist in small isolated populations, and closely related yet outbred Western massasauga rattlesnakes (Sistrurus tergeminus). In terms of inbreeding,S. catenatusgenomes had a greater number of runs of homozygosity of varying sizes, indicating sustained inbreeding through repeated bottlenecks when compared toS. tergeminus. At the species level, outbredS. tergeminushad higher genome‐wide levels of mutation load in the form of greater numbers of derived deleterious mutations compared toS. catenatus, presumably due to long‐term purging of deleterious mutations inS. catenatus. In contrast, mutations that escaped species‐level drift effects withinS. catenatuspopulations were in general more frequent and more often found in homozygous genotypes than inS. tergeminus, suggesting a reduced efficiency of purifying selection in smallerS. catenatuspopulations for most mutations. Our results support an emerging idea that the historical demography of a threatened species has a significant impact on the type of genetic load present, which impacts implementation of conservation actions such as genetic rescue.

 

Managing endangered species in fragmented landscapes requires estimating dispersal rates between populations over contemporary timescales. Here, we developed a new method for quantifying recent dispersal using genetic pedigree data for close and distant kin. Specifically, we describe an approach that infers missing shared ancestors between pairs of kin in habitat patches across a fragmented landscape. We then applied a stepping‐stone model to assign unsampled individuals in the pedigree to probable locations based on minimizing the number of movements required to produce the observed locations in sampled kin pairs. Finally, we used all pairs of reconstructed parent‐offspring sets to estimate dispersal rates between habitat patches under a Bayesian model. Our approach measures connectivity over the timescale represented by the small number of generations contained within the pedigree and so is appropriate for estimating the impacts of recent habitat changes due to human activity. We used our method to estimate recent movement between newly discovered populations of threatened Eastern Massasauga rattlesnakes (Sistrurus catenatus) using data from 2996 RAD‐based genetic loci. Our pedigree analyses found no evidence for contemporary connectivity between five genetic groups, but, as validation of our approach, showed high dispersal rates between sample sites within a single genetic cluster. We conclude that these five genetic clusters of Eastern Massasauga rattlesnakes have small numbers of resident snakes and are demographically isolated conservation units. More broadly, our methodology can be widely applied to determine contemporary connectivity rates, independent of bias from shared genetic similarity due to ancestry that impacts other approaches.

 

An important goal of conservation genetics is to determine if the viability of small populations is reduced by a loss of adaptive variation due to genetic drift. Here, we assessed the impact of drift and selection on direct measures of adaptive variation (toxin loci encoding venom proteins) in the eastern massasauga rattlesnake (Sistrurus catenatus), a threatened reptile that exists in small isolated populations. We estimated levels of individual polymorphism in 46 toxin loci and 1,467 control loci across 12 populations of this species, and compared the results with patterns of selection on the same loci following speciation ofS. catenatusand its closest relative, the western massasauga (S. tergeminus). Multiple lines of evidence suggest that both drift and selection have had observable impacts on standing adaptive variation. In support of drift effects, we found little evidence for selection on toxin variation within populations and a significant positive relationship between current levels of adaptive variation and long‐ and short‐term estimates of effective population size. However, we also observed levels of directional selection on toxin loci among populations that are broadly similar to patterns predicted from interspecific selection analyses that pre‐date the effects of recent drift, and that functional variation in these loci persists despite small short‐term effective sizes. This suggests that much of the adaptive variation present in populations may represent an example of “drift debt,” a nonequilibrium state where present‐day levels of variation overestimate the amount of functional genetic diversity present in future populations.

 

Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long‐sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins (“venom interactive proteins” [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum‐based resistance is both population‐ and species‐specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum “resistome” involves broad‐based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co‐evolutionary interactions between species at the molecular level.

 

Abstract Understanding the joint roles of protein sequence variation and differential expression during adaptive evolution is a fundamental, yet largely unrealized goal of evolutionary biology. Here, we use phylogenetic path analysis to analyze a comprehensive venom-gland transcriptome dataset spanning three genera of pitvipers to identify the functional genetic basis of a key adaptation (venom complexity) linked to diet breadth (DB). The analysis of gene-family-specific patterns reveals that, for genes encoding two of the most important venom proteins (snake venom metalloproteases and snake venom serine proteases), there are direct, positive relationships between sequence diversity (SD), expression diversity (ED), and increased DB. Further analysis of gene-family diversification for these proteins showed no constraint on how individual lineages achieved toxin gene SD in terms of the patterns of paralog diversification. In contrast, another major venom protein family (PLA2s) showed no relationship between venom molecular diversity and DB. Additional analyses suggest that other molecular mechanisms—such as higher absolute levels of expression—are responsible for diet adaptation involving these venom proteins. Broadly, our findings argue that functional diversity generated through sequence and expression variations jointly determine adaptation in the key components of pitviper venoms, which mediate complex molecular interactions between the snakes and their prey. 

Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators. Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats (forest, pasture, degraded area, and floodplain) in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey. 

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome ofBothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A₂(PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option.

 

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species’ divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

 

Identifying the environmental correlates of divergence in functional traits between populations can provide insights into the evolutionary mechanisms that generate local adaptation. Here, we assess patterns of population differentiation in expressed venom proteins in Northern Pacific rattlesnakes (Crotalus oreganus) from 13 locations across California. We evaluate the relative importance of major biotic (prey species community composition), abiotic (temperature, precipitation, and elevation) and genetic factors (genetic distance based on RADseq loci) as correlates of population divergence in venom phenotypes. We found that over half of the variation in venom composition is associated with among-population differentiation for genetic and environmental variables, and that this variation occurred along axes defining previously observed functional trade-offs between venom proteins that have neurotoxic, myotoxic and hemorrhagic effects. Surprisingly, genetic differentiation among populations was the best predictor of venom divergence, accounting for 46% of overall variation, whereas differences in prey community composition and abiotic factors explained smaller amounts of variation (23% and 19%, respectively). The association between genetic differentiation and venom composition could be due to an isolation-by-distance effect or, more likely, an isolation-by-environment effect where selection against recent migrants is strong, producing a correlation between neutral genetic differentiation and venom differentiation. Our findings suggest that even coarse estimates of prey community composition can be useful in understanding the selection pressures acting on patterns of venom protein expression. Additionally, our results suggest that factors other than adaptation to spatial variation in prey need to be considered when explaining population divergence in venom.